ASTM D6503-99(2009)

NOTICE: This standard has either been superseded and replaced by a new version or withdrawn.
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Designation: D6503 − 99(Reapproved 2009)
Standard Test Method for
1,2
Enterococci in Water Using Enterolert
This standard is issued under the fixed designation D6503; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision.Anumber in parentheses indicates the year of last reapproval.A
superscript epsilon (´) indicates an editorial change since the last revision or reapproval.
1. Scope 3.2 Definitions of Terms Specific to This Standard:
3.2.1 enterococci, n—a gram positive bacteria possessing
1.1 This test method covers a simple procedure for the
the enzyme β-D-glucosidase, which cleaves the nutrient indi-
detectionofenterococciinwaterandwastewater.Itisbasedon
catorandproducesfluorescenceunderalongwavelength(366
IDEXX’spatentedDefinedSubstrateTechnology(DST). This
nm) ultraviolet (UV) light.
product, Enterolert, utilizes a nutrient indicator that fluoresces
when metabolized. It can detect these bacteria at one colony 3.2.2 most probable number (MPN), n—a statistical method
for determining bacterial density based on the Poisson distri-
forming unit (CFU)/100 mL within 24 h. The presence of this
microorganisminwaterisanindicationoffecalcontamination bution.
and the possible presence of enteric pathogens.
3.2.3 presence-absence, n—a term used to indicate if en-
terococci is present in a water sample. It is a qualitative value,
1.2 This test method can be used successfully with drinking
“yes” or “no” for reporting results.
water, source water, recreational (fresh and marine) water, and
bottled water. It is the user’s responsibility to ensure the
3.2.4 quanti-tray , n—a system for the quantification of
validity of this test method for waters of untested matrices.
enterococci. It consists of a sealer and trays which have
multi-wells and can enumerate up to 2000 CFU/100 mL
1.3 The values stated in SI units are to be regarded as
without dilution.
standard. No other units of measurement are included in this
standard.
3.2.5 snap pack, n—apackagecontainingEnterolertreagent
for testing 100-mL sample either in the P/A format or
1.4 This standard does not purport to address all of the
quantitatively, that is, Quanti-Tray system).
safety concerns, if any, associated with its use. It is the
responsibility of the user of this standard to establish appro-
4. Summary of Test Method
priate safety and health practices and determine the applica-
bility of regulatory limitations prior to use.
4.1 Thistestmethodisusedforthedetectionofenterococci,
such as E. faecium, E. faecalis in drinking water, source water,
2. Referenced Documents
recreationalwaters(marinewaterandfresh),andbottledwater.
2.1 ASTM Standards: Whenthereagentisaddedtothesampleandincubatedat41 6
D1129Terminology Relating to Water
0.5°C for 24 h, Enterolert can detect these bacteria at 1
D1193Specification for Reagent Water CFU/100 mL. Fluorescence is produced when enterococci
D2777Practice for Determination of Precision and Bias of
metabolizes the nutrient indicator. Enterolert can be used as a
Applicable Test Methods of Committee D19 on Water presence-absence test or for quantification (5-tube, 10-tube
D3370Practices for Sampling Water from Closed Conduits
MPN, 15-tube serial dilution or the Quanti-Tray system).
3. Terminology
5. Significance and Use
3.1 Definitions—For definitions of terms used in this test
5.1 This test provides an easy and reliable method for the
method, refer to Terminology D1129.
detection of enterococci in water within 24 h. For recreational
water (fresh and marine) testing is performed to insure areas
are safe for swimming. Enterolert also can be used for testing
This test method is under the jurisdiction ofASTM Committee D19 on Water
bottled water and drinking water.
and is the direct responsibility of Subcommittee D19.24 on Water Microbiology.
Current edition approved May 1, 2009. Published June 2009. Originally
approved in 1999. Last previous edition approved in 2005 as D6503–99 (2005).
6. Interferences
DOI: 10.1520/D6503-99R09.
Trademark of IDEXX Laboratories, One Idexx Dr., Westbrook, ME 04092.
6.1 The presence of Bacillus spp. can interfere with the
For referenced ASTM standards, visit the ASTM website, www.astm.org, or
testing of marine water samples. To eliminate interference, a
contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM
1:10 dilution is required with sterile water (deionized or
Standards volume information, refer to the standard’s Document Summary page on
the ASTM website. distilled).
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
D6503 − 99 (2009)
7. Apparatus 11.2.4 Follow the Enterolert presence/absence steps listed
above to test these controls. Compare the test results to the
7.1 Ultraviolet Lamp, 6-watt long wavelength (366 nm).
following expected results:
7.2 41°C Incubator (60.5°C), air or water bath.
Control ATTC No. Expected Result
Enterococcus faecium 335667 Fluorescence
7.3 Vessels, sterile, nonfluorescent.
Serratia marcescens (g, –) 43862 No fluorescence
7.4 Quanti-Tray Sealer. Aerococcus viridians (g, +) 10400 No fluorescence
7.5 Quanti-Tray or Quanti-Tray 2000.
12. Procedure
12.1 Presence/Absence—See package insert.
8. Reagents and Materials
12.1.1 Samples should be brought to room temperature (18
8.1 Purity of Water—Unless otherwise indicated, references
to 30°C).
towatershallbeunderstoodtomeanreagentwaterconforming
12.1.2 Carefully separate one snap pack from the strip.
to Specification D1193, Type IV. Sterilize the water by either
12.1.3 Tap the snap pack to insure that all of the powder is
autoclaving or by sterile filtration (0.22 micron-filtered water).
towards the bottom of the pack.
8.2 Enterolert Test Kit.
12.1.4 Open the pack by snapping back the top of the score
line. Do not touch the opening of pack.
9. Precautions
12.1.5 Add the reagent to a 100-mLwater sample, which is
9.1 The analyst must observe the normal good laboratory
in a sterile, transparent, nonfluorescent vessel.
practices and safety procedures required in a microbiology
12.1.6 Aseptically cap and seal the vessel.
laboratory while preparing, using, and disposing of cultures,
12.1.7 Shake until dissolved.
reagents and materials and while operating sterilization equip-
12.1.8 Incubate Enterolert for 24 h at 41 6 0.5°C,
ment and other equipment.
12.1.9 Read results at 24 h. If the sample is inadvertently
incubated over 28 h without observation, the following guide-
10. Sampling
lines apply: Lack of fluorescence after 28 h is a valid negative
10.1 CollectthesampleasdescribedindetailintheUSEPA test. Fluorescence after 28 h is an invalid result.
microbiological methods manual and in accordance with 12.1.10 Check for fluorescence by placing a 6-W 366-nm
Practices D3370. UV light within 5 in. of the sample in a dark environment. Be
sure the light is facing away from your eyes and towards the
10.2 Sample Storage Temperature and Handling
vessel. If fluorescence is observed, the presence of enterococci
Conditions—Ice or refrigerate water samples at a temperature
is confirmed.
of 2 to 8°C during transit to the laboratory. Use insulated
containers to ensure proper maintenance of storage tempera- 12.2 MPN—Quanti-tray enumeration test procedure for
tures.Take care that sample bottles are not totally immersed in
100-mL sample (see package insert).
water during transit or storage. 12.2.1 Follow steps 12.1.1-12.1.7.
12.2.2 Pour the reagent sample into the Quanti-Tray avoid-
10.3 Holding Time Limitations—Examine samples, as soon
ing contact with the foil tab and seal the tray according to the
as possible, after collection. Do not hold samples longer than
Quanti-Tray package insert.
6h between collection and initiation of analyses.
12.2.3 Incubate for 24 h at 41 6 0.5°C.
12.2.4 Follow the same interpretation instructions from
11. Quality Control Check
12.1.9through12.1.10,andcountthenumberofpositivewells.
11.1 Check and record temperatures in incubators daily to
Refer to the MPN table (see Table 1) provided with the
ensure temperature is within stated limits.
Quanti-Tray to determine the CFU/100 mL.
11.2 Qualitycontrolshouldbeconductedoneachnewlotof
12.3 MPN—5-tube × 20 mL, 10-tube × 10 mL and 15-tube
Enterolert. See package insert for the recommended quality
serial dilution.
control procedure, which consists of the following protocol:
12.3.1 Follow 12.1.1-12.1.7.
11.2.1 For each type of theAmerican Type Culture Collec-
12.3.2 sterile nonfluorescent tubes or transfer 20 mLof the
tion (ATCC) bacterial strain listed below, streak the culture
reagent sample into five sterile nonfluorescent tubes.
ontolabeledTSAorbloodagarplatesandincubateat35°Cfor
12.3.3 Incubate for 24 h at 41 6 0.5°C.
18 to 24 h.
12.3.4 Follow 12.1.9 and 12.1.10 for interpretation.
11.2.2 For each bacterial strain, touch a 1-µl loop to a
12.3.5 Refer to the MPN tables (see Tables 2-4) to deter-
colony and use it to inoculate a labeled test tube containing
mine the CFU/100 mL.
5mL of sterile deionized water. Close cap and shake thor-
oughly.
13. Calculation
11.2.3 For each bacterial strain, take a 1-µl loop from the
13.1 For P/A, there are no calculations. For quantification,
test tube and use it to inoculate a labeled vessel containing
refer to Quanti-Tray MPN tables and for the 5, 10, and 15 tube
100mL.
test results refer to the respective MPN tables.
Bordner, R.H.,Winter, J.A., and Scarpino, P.V., Eds., Microbiological Methods
for Monitoring the Environment, Water, and Wastes, EPA-600/8-78-017. Standard Methods for the Examination of Water and Waste Water,19thEdition.
D6503 − 99 (2009)
TABLE 1 51-Well Quanti-Tray MPN Table
95 % Confidence Limits
No. of Wells Giving
MPN/100-mL Sample
Positive Reaction
Lower Upper
0 <1 0.0 3.7
1 1.0 0.3 5.6
2 2.0 0.6 7.3
3 3.1 1.1 9.0
4 4.2 1.7 10.7
5 5.3 2.3 12.3
6 6.4 3.0 13.9
7 7.5 3.7 15.5
8 8.7 4.5 17.1
9 9.9 5.3 18.8
10 11.1 6.1 20.5
11 12.4 7.0 22.1
12 13.7 7.9 23.9
13 15.0 8.8 25.7
14 16.4 9.8 27.5
15 17.8 10.8 29.4
16 19.2 11.9 31.3
17 20.7 13.0 33.3
18 22.2 14.1 35.2
19 23.8 15.3 37.3
20 25.4 16.5 39.4
21 27.1 17.7 41.6
22 28.8 19.0 43.9
23 30.6 20.4 46.3
24 32.4 21.8 48.7
25 34.4 23.3 51.2
26 36.4 24.7 53.9
27 38.4 26.4 56.6
28 40.6 28.0 59.5
29 42.9 29.7 62.5
30 45.3 31.5 65.6
31 47.8 33.4 69.0
32 50.4 35.4 72.5
33 53.1 37.5 76.2
34 56.0 39.7 80.1
35 59.1 42.0 84.4
36 62.4 44.6 88.8
37 65.9 47.2 93.7
38 69.7 50.0 99.0
39 73.8 53.1 104.8
40 78.2 56.4 111.2
41 83.1 59.9 118.3
42 88.5 63.9 126.2
43 94.5 68.2 135.4
44 101.3 73.1 146.0
45 109.1 78.6 158.7
46 118.4 85.0 174.5
47 129.8 92.7 195.0
48 144.5 102.3 224.1
49 165.2 115.2 272.2
50 200.5 135.8 387.6
51 >200.5 146.1 infinite
14. Report different matrixes at three levels following Practice D2777.
Outliers were rejected in accordance with the statistical tests
14.1 Report as positive or negative for presence/absence
outlined in Practice D2777. All data from one technician was
testing.
rejected for recreational water-marine and single values were
14.2 Reporting of results is based on calculation of entero-
rejected for both recreational water-fresh at the low level and
cocci density determined from the appropriate MPN tables.
forrecreationalwater-marineatthelowlevel.Themeancount,
15. Precision and Bias
the overall standard deviation (St), and the single operator
standard deviation (so), are indicated in Table 5.
15.1 Precision—A limited collaborative study was con-
ducted. Nine technicians from three laboratories tested three
15.2 Bias—Themeanvalueobtainedforthesamples(drink-
ing water, recreational water fresh and marine) from the nine
Supporting data have been filed atASTM International Headquarters and may
technicians for the low-, mid- and high-spiked samples all fall
be obtained by requesting Research Report RR:D19-1167.
D6503 − 99 (2009)
TABLE 2 IDEXX Quanti-Tray/2000 MPN Table (cfu/100 mL)
No. Large No. Small Wells Positive
Wells 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23
Positive
0 <1 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 15.1 16.1 17.1 18.1 19.1 20.2 21.2 22.2 23.2
1 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.1 9.1 10.1 11.1 12.1 13.2 14.2 15.2 16.2 17.3 18.3 19.3 20.4 21.4 22.4 23.5 24.5
2 2.0 3.0 4.1 5.1 6.1 7.1 8.1 9.2 10.2 11.2 12.2 13.3 14.3 15.3 16.4 17.4 18.5 19.5 20.6 21.6 22.6 23.7 24.8 25.8
3 3.1 4.1 5.1 6.1 7.2 8.2 9.2 10.3 11.3 12.4 13.4 14.4 15.5 16.5 17.6 18.6 19.7 20.8 21.8 22.9 23.9 25.0 26.1 27.1
4 4.1 5.2 6.2 7.2 8.3 9.3 10.4 11.4 12.5 13.5 14.6 15.6 16.7 17.8 18.8 19.9 21.0 22.0 23.1 24.2 25.2 26.3 27.4 28.5
5 5.2 6.3 7.3 8.4 9.4 10.5 11.5 12.6 13.7 14.7 15.8 16.9 17.9 19.0 20.1 21.2 22.2 23.3 24.4 25.5 26.6 27.7 28.8 29.9
6 6.3 7.4 8.4 9.5 10.6 11.6 12.7 13.8 14.9 15.9 17.0 18.1 19.2 20.3 21.4 22.5 23.6 24.7 25.8 26.9 28.0 29.1 30.2 31.3
7 7.4 8.5 9.6 10.7 11.8 12.8 13.9 15.0 16.1 17.2 18.3 19.4 20.5 21.6 22.7 23.8 24.9 26.0 27.1 28.3 29.4 30.5 31.6 32.8
8 8.6 9.7 10.8 11.9 13.0 14.1 15.2 16.3 17.4 18.5 19.6 20.7 21.8 22.9 24.1 25.2 26.3 27.4 28.6 29.7 30.8 32.0 33.1 34.3
9 9.8 10.9 12.0 13.1 14.2 15.3 16.4 17.5 18.7 19.8 20.9 22.0 23.2 24.3 25.4 26.6 27.7 28.9 30.0 31.2 32.3 33.5 34.6 35.8
10 11.0 12.1 13.2 14.3 15.5 16.6 17.7 18.9 20.0 21.1 22.3 23.4 24.6 25.7 26.9 28.0 29.2 30.3 31.5 32.7 33.8 35.0 36.2 37.4
11 12.2 13.4 14.5 15.6 16.8 17.9 19.1 20.2 21.4 22.5 23.7 24.8 26.0 27.2 28.3 29.5 30.7 31.9 33.0 34.2 35.4 36.6 37.8 39.0
12 13.5 14.6 15.8 16.9 18.1 19.3 20.4 21.6 22.7 23.9 25.1 26.3 27.5 28.6 29.8 31.0 32.2 33.4 34.6 35.8 37.0 38.2 39.4 40.7
13 14.8 16.0 17.1 18.3 19.5 20.6 21.8 23.0 24.2 25.4 26.6 27.8 29.0 30.2 31.4 32.6 33.8 35.0 36.2 37.5 38.7 39.9 41.1 42.4
14 16.1 17.3 18.5 19.7 20.9 22.1 23.3 24.4 25.7 26.9 28.1 29.3 30.5 31.7 33.0 34.2 35.4 36.7 37.9 39.1 40.4 41.6 42.9 44.2
15 17.5 18.7 19.9 21.1 22.3 23.5 24.7 25.9 27.2 28.4 29.6 30.9 32.1 33.3 34.6 35.8 37.1 38.4 39.6 40.9 42.2 43.4 44.7 46.0
16 18.9 20.1 21.3 22.6 23.8 25.0 26.2 27.5 28.7 30.0 31.2 32.5 33.7 35.0 36.3 37.5 38.8 40.1 41.4 42.7 44.0 45.3 46.6 47.9
17 20.3 21.6 22.8 24.0 25.3 26.5 27.8 29.1 30.3 31.6 32.9 34.1 35.4 36.7 38.0 39.3 40.6 41.9 43.2 44.5 45.9 47.2 48.5 49.8
18 21.8 23.1 24.3 25.6 26.9 28.1 29.4 30.7 32.0 33.3 34.6 35.9 37.2 38.5 39.8 41.1 42.4 43.8 45.1 46.4 47.8 49.1 50.5 51.9
19 23.3 24.6 25.9 27.2 28.5 29.8 31.1 32.4 33.7 35.0 36.3 37.6 39.0 40.3 41.6 43.0 44.3 45.7 47.1 48.4 49.8 51.2 52.6 54.0
20 24.9 26.2 27.5 28.8 30.1 31.4 32.8 34.1 35.4 36.8 38.1 39.5 40.8 42.2 43.6 44.9 46.3 47.7 49.1 50.5 51.9 53.3 54.7 56.1
21 26.5 27.8 29.2 30.5 31.8 33.2 34.5 35.9 37.3 38.6 40.0 41.4 42.8 44.1 45.5 46.9 48.4 49.8 51.2 52.6 54.1 55.5
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